Although data from serum lipid composition suggest that a potential major source of brain PUFA is lipoprotein-associated complex
lipids, few studies have as yet addressed specific details of PUFA transport and uptake by this mechanism. Numerous lipoprotein
classes and glycerolipid pools may be involved. Thus, investigations directed at the identification and functional characterization
of PUFA-selective lipoprotein classes and PUFA-enriched glycerolipid pools would be enlightening. It may be possible to utilize
stable PUFA isotopes (13C or perdeuterated) to identify and follow these lipoproteins/glycerolipids in feeding studies.

Role of barrier cells and astrocytes in the uptake and processing of PUFA or lipoprotein-associated PUFA

Studies that parallel the focus on serum lipid composition (particularly lipoproteins) should also be directed at cells carrying
out barrier functions between blood and neurons. Foremost among these barrier cells are cerebral endothelium of the blood-brain
barrier, choroid plexus epithelium that produce the bulk of cerebrospinal fluid, and astrocytes that closely associate with
both cerebral blood vessels and neurons. The distribution, molecular nature, and function of lipoprotein lipase, lipoprotein
receptors, and intracellular mechanisms for lipoprotein processing or trafficking in these cells are some of the major areas
that require attention.

Once PUFAs are free of their carrier proteins and/or glycerolipids, a different group of binding and transport proteins are
likely to be important in directing the flow of PUFA across barrier cells or within the brain. In this regard molecular and
cell biological approaches should be aimed at understanding how fatty acid binding proteins (FABP) and fatty acid transport
proteins (FATP) are distributed and how they function. Attention should also be directed at the specificity of enzymes that
esterify PUFA, since the selectivity observed in membrane lipids may be regulated significantly at the esterification level.

Finally, additional carrier proteins or glycerolipids are likely to be involved in the intercellular distribution of PUFA
within the brain parenchyma. For example, astrocytes are known to be a source of apolipoprotein synthesis in the brain, but
little is known about the function of brain apolipoproteins in the intercellular exchange of PUFA. The fact that the E4 allele
of apolipoprotein E is a major risk factor for Alzheimer's disease provides a significant allure to this area of study.

Local synthesis of PUFA

Although there is strong evidence for local synthesis of long-chain PUFA by brain-derived cells, recent advances in the molecular
and cell biology of desaturase enzymes and peroxisomal assembly have yet to be applied systematically to the brain. The application
of this new knowledge, particularly using genetic engineering approaches, should provide the clearest

evidence yet on the role of local synthesis in the accretion of PUFA by brain. It might also provide a basis for developing
molecular approaches to therapy in neurological disorders where long-chain PUFAs are deficient.

The method, model and "operational equations" presented at the workshop, for quantifying in vivo brain turnover rates and half-lives of fatty acids during uptake from plasma to the brain, can be applied to the elucidation
of uptake rates of nutritionally -provided omega-6 PUFA (e.g. linoleic acid, LA; gamma-linolenic acid, GLA, arachidonic acid,
AA), omega-3 PUFA (e.g. alpha-linolenic acid, ALA; eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA) and of dietary
saturated and monounsaturated fatty acids. In the case of saturated and monounsaturated fatty acids, studies could help clarify
the possible differences between their uptake versus de novo synthesis in the developing and in the adult brains.

Effects of centrally acting drugs on the steady state of fatty acids

The above approach can also be applied to the assessment of changes in steady states in response to centrally acting drugs
and pathological changes in bipolar and other disorders. When combined with neuroimaging intravenously injected radiolabeled
PUFA can also be utilized to examine neuroplastic remodeling of brain fatty acids and lipid membranes.

Role of plasmalogens in glial and neuronal tissue

It was noted that AA- and DHA-containing plasmalogens (the glycerophospholipids containing an enol-ether group at the sn-1
position) play important roles as potential protectors of neurons from oxidative stress and suppliers of free AA and DHA to
the cell. They can function also as modulators of neuronal membrane physical state and permeability both in the developing
and in the mature brain. Plasmalogens are formed in peroxisomes, therefore they could be involved in a wide range of pathological
states such as peroxisomal biogenesis disorders (Zellweger's syndrome and neonatal leukodystrophy) as well as ischemia or
neurodegerative diseases, such as Alzheimer's disease, and even spinal cord trauma. All of these issues warrant rapid further
elucidation.

It would also be important to understand the contribution of plasmalogens to the free fatty acid pool and therefore their
contributions to prostaglandin-mediated processes in the developing and mature neuron in health and disease.

The role of DHA in the developing and adult brain

Results reported in Session 2 and 3 settle neither the question whether saturated and monounsaturated fatty acids are taken
up by the brain or whether they are synthesized de novo by the brain, nor the question whether developing brain is similar or identical with adult brain in this regard. Studies
on the gene regulatory effects of EPA and DHA in the liver and other organs on lipogenic enzymes do not extend to the brain
in a satisfactory fashion. A strong need exists in elucidating the molecular biology and genetics of brain lipogenesis and
its regulation by long-chain, omega-3 fatty acids (EPA and DHA) in both the developing and the adult brain.

The accelerated in utero uptake of DHA (by the rat embryo) appears characteristic both to animals and humans. Since the mother is the major supplier
of DHA to the developing rat brain, a low circulating level of plasma DHA in the mother could lower the embryo's DHA brain
levels. Direct intra-amniotic injection of DHA ethyl ester restores embryonic brain uptake of DHA to normal levels. Additional
research into the mechanisms of DHA accretion and depletion in embryonic and developing rat brain and into the potential behavioral
or cognitive consequences of below normal DHA levels in the embryonic and postnatal brain should shed light on why DHA and
possibly AA supplementation of infant formulas would be important for normal brain development. Elucidating the consequences
of DHA deficiency during the developmental period is especially important due to the reported potential role of DHA to protect
the developing brain from oxidative stress.

The extension of these in vivo studies to humans would render the development of appropriate non-invasive techniques for quantitation of neuronal changes
due to DHA presence or absence including, but not limited to the development of new imaging and tracer approaches highly desirable.

The question of the role of DHA in the management of environmental stress and oxidative stress in the adult brain is also
worthy of exploration.

A major breakthrough in understanding the role of DHA in signal transduction was recently reported in the outer rod segments
(ROS) of the retina. It appears that the role of DHA in ROS membranes is attributable to its six double bonds that provide
and ensure a "most favored" microenvironment in which rhodopsin can absorb a photon of light and undergo the rapid conformational
changes that result in the activation of a G-protein. This activation initiates the chain of biochemical events in the visual
transduction pathway that ultimately leads to hyperpolarization of the plasma membrane. This observation, based on solid methodological
determinations indicates that the high concentrations of DHA in the rhodopsin the outer rod segments might be of paramount
importance to efficient signal transduction. DHA might have similar functions in the transduction of the neuronal signals
through the membrane and synapses. Validating this potential role of DHA in the different neurons of different areas of the
brain and should contribute significantly to our understanding of the functioning of the central nervous system.

It is also important to understand what implications does DHA deficiency, during brain development, have on neuronal signal
transduction and transmission.

Intermediary metabolism of PUFA and brain disorders

Answers should be sought to questions involving post-uptake shuttling and metabolism of AA, EPA and DHA. Such questions are
exemplified in the following: How does DHA, taken up by the brain through the BBB or synthesized from its precursors by astrocytic
peroxisomes, reach its neuronal locations? Are there special DHA transporters in the brain? Are astrocytes mostly devoid of
DHA and function primarily as elongation/ desaturation tools of EPA and its higher homolog docosapentaenoic acid (DPA)? What are the functions of EPA and DPA in astrocytes?
Are EPA and DHA naturally segregated between astrocytes (EPA) and neurons (DHA)? What are the eicosanoids produced from EPA
and what are their functions in the brain? What are docosanoids and what are their functions in the brain? Are these functions
related to the pathophysiology of neuropsychobehavioral disorders such as: bipolar disorder, unipolar depression and schizophrenia?
Do EPA, DPA and DHA exert the same therapeutic effect in these disorders? Etc.

DHA, EPA and apoptosis of glial and neuronal cells

-The finding that DHA can potentially inhibit neuronal apoptosis in two cell lines and that this inhibitory effect appears
related to an increase in PS concentration raises several questions on yet another potential physiological role of DHA namely
that of long-term survival of the neuronal cell. Does depletion of DHA result in neuronal death? If that effect of DHA is
proven, is this effect reversible? Is there a connection between DHA depletion and the pathophysiology of inherited neurodegenerative
diseases or Alzheimer's disease? Is the anti-apoptotic effect of DHA responsible for its apparent segregation to the neuron?
What would be the consequences of long-term DHA storage in astrocytes, which appear to eliminate free DHA as it is formed
from its precursors. How would the anti-apoptotic effect persist in animal models? All of these questions deserve to be answered.

Therapeutic trials in peroxisomal disorders will form an increasingly important future priority. This represents an obvious
priority, since these disorders cause such severe disability. In addition, the tools to carry out and evaluate therapies are
becoming available to an increasing extent. The existence of animal models is a key feature, which opens also the possibility
of evaluating in utero therapy. Another key tool is the increasing availability of neuroimaging studies, such as MRI, magnetic resonance spectroscopy
and diffusion fiber tracking techniques. Improved understanding of the molecular and enzymatic defects has led to new therapeutic
approaches, of which DHA therapy of peroxisomal biogenesis disorders (PBD) is a prime example. Evaluation of therapeutic interventions
in peroxisomal disorders is complicated by the marked and only partially predictable variability in natural history, and also
the desirability for early initiation of therapy at time before irreversible damage has occurred. Discussion took place about
the use of randomized placebo-controlled studies. Some participants took the position that such trials were not ethically
justifiable in circumstances when the consequences of the disease are severe and the therapy is essentially free of side effects,
as is the case with DHA. Others took the position that carefully designed placebo controlled trials supervised by an independent
treatment effects monitoring committee represented the most rapid method of determining effectiveness and safety of therapy
and could be conducted in a way that safeguarded the best interests of the patients and their families.

The application and evaluation of DHA therapy in PBD disorders exemplifies another important principle and opportunity. PBD
patients (and animal models of these disorders) have profound disturbances in DHA metabolism and they show profound neurologic
deficits. This combination provides the opportunity to determine relatively quickly the extent to which DHA therapy can be
of benefit and when and how it should be administered. However, there is increasing evidence that more subtle DHA deficits,
traceable in most instances to environmental circumstances that could be altered, are of clinical significance in much larger
groups of individuals.

Understanding the functions of fatty acids in peroxisomal biogenesis disorders

The study of fatty acids in PBD disorders represents an important and promising field of investigation. The PBD disorders
are associated with characteristic and severe handicaps and can be diagnosed early, including prenatally, by non-invasive
and reliable diagnostic assays. Characteristic and striking abnormalities in fatty acid profiles and metabolism are present
in all of these disorders. Some of these abnormalities can be normalized completely or in part, by dietary manipulations or
by the administration of non-toxic natural compounds. Results on improvement in myelination of Zellweger patients following
DHA supplementation therapy, over time, raises a slew of new questions. What is the mechanism by which DHA supplementation
improves myelination? Since DHA is not abundant in white matter, is DHA present in oligodendrocytes where it exerts its effect
indirectly through correction of reduced levels? Or rather does DHA lower VLCFA levels (which have been implicated in demyelination)?
Does DHA raise plasmalogen levels known to be low in Zellweger syndrome thus protecting membranes from oxidative stress (see
also recommendations in the March 4, 2000 Morning Session)? Animal models for most of these disorders are now available and
new ones can be developed as needed.

Animal models for study of fatty acid function in glia and neurons

A variety of future research programs flow naturally from these observations, and several are already in progress. Many of
the PBD patients, as well as the animal models, show characteristic defects of neuronal migration which take place during
fetal life. Gaining an understanding of the mechanisms that lead to disturbances in this fundamental biological process may
lead to therapies that can be applied during early phases of development, possibly even during fetal life, and will also contribute
to an understanding of normal brain development. The availability of a mouse model that displays similar neuronal migration
defects is of immense value here. It is likely that a variety of models can be developed in which genetic defects are more
restricted and targeted and that these will permit delineation of the comparative roles of specific genetic and biochemical
defects. Other neuropathological changes occur post-developmentally. Examples of these are changes secondary to the accumulation
of branched chain fatty acids such as phytanic acid, since the abnormal accumulation of this substance does not commence until
after birth. The most striking example of post-developmental pathology is the distal axonopathy that occurs in adults with
AMN, and appears attributable in some way to the accumulation of very long chain fatty acids.

The studies in rodents who have been deprived of DHA for three generations exemplify the broad implications of this research
approach and provide recommendations for future research. These studies have shown that DHA deprivation in rodents impairs
spatial tasks and olfactory discrimination. Studies of behavioral tasks that are cued to olfactory modality are an ideal manner
to examine higher levels of learning/memory related performance in rodents because they are macrosomatic. The reversal learning
task may prove a useful approach for the study of DHA adequacy, and may prove of great value for the definition of the role
of DHA in normal human brain development.

Omega-3 PUFA and the hepatic side of Zellweger syndrome

The peroxisomal defects of the Zellweger brain extend to the liver and kidney. The liver is known to be an important location
for elongation and desaturation of alpha-linolenic acid (ALA) and other intermediates in the pathway to EPA and DHA. Several
issues deserve further studies. For example: Is ALA elongated and desaturated in the livers of Zellweger patients? Is there
partial production of DHA in the liver but not sufficient for satisfying needs? Can the existing animal models of Zellweger
syndrome be used to develop methods that will allow a comparative assessment of plasma, erythrocyte and brain levels of EPA
and DHA of healthy individuals and of Zellweger animal models? Could such methods be extended to other diseases of the central
nervous system?